These features are, therefore, radically different from those of CLL and other B-cell tumors for which we observe a more heterogeneous distribution pattern reflecting major pathogenetic and clinical differences

These features are, therefore, radically different from those of CLL and other B-cell tumors for which we observe a more heterogeneous distribution pattern reflecting major pathogenetic and clinical differences.3,4,13,14Lengths of HCDR3 AA sequences in MM SX-3228 were normally distributed with a mean value of 15.5 AA (range 629, SD 3.89), in accordance with published series of healthy PC (Figure 3B). == Physique 3. == Analysis of multiple myeloma immunoglobulin repertoire does not support a pathogenetic role for antigen selection in this tumor. Keywords:multiple myeloma, immunoglobulin, antigen SX-3228 selection, heavy chain gene == Introduction == Characterization of the immunoglobulin receptor has provided useful insights into the pathogenesis and natural history of lymphoid neoplasms.17In particular, large databases of immunoglobulin heavy chain gene (IGH) sequences have been analyzed to demonstrate the role of antigen stimulation as a key environmental driver of malignant transformation. The recognition of stereotyped clusters of immunoglobulin receptors in chronic lymphocytic leukemia (CLL) provides strong evidence to support the role of antigen-driven mechanisms in the pathogenesis of this neoplasm.812Furthermore, recent reports in mantle cell lymphoma (MCL)13and marginal zone lymphoma (MZL)14have led to speculation that stereotyped clusters of immunoglobulin receptors might SX-3228 represent a common phenomenon across most, if not all, mature B-cell tumors. Although terminally-differentiated malignant plasma cells (PC) do not express immunoglobulin receptors, it is known that myelomagenesis is usually a complex multi-step process that might involve earlier B-cell precursors.15In particular, the class switch recombination mechanisms involved in multiple myeloma (MM) related chromosomal translocations suggest that early events in the development of MM occur during germinal center (GC) maturation.16Furthermore, the presence of pre-switch B cells idio-typically related to malignant PC has been reported.17Therefore, there is a clear rationale to evaluate the MM IGH repertoire and to search for stereotyped clusters of immunoglobulin receptors on appropriately sized patient series. This will allow us to investigate further into the role of antigen-driven stimulation in B-lymphoid tumorigenesis and whether antigen stimulation might play a role in early myelomagenesis. Published databases of IGH MM sequences1,6,7each include less than 80 evaluable patients and are, therefore, too small to perform extensive cluster analysis of immunoglobulin receptors. We have combined a large number of MM IGH sequences obtained at our institutions for minimal residual disease (MRD) evaluation18with all the sequences reported in the literature, leading to a panel of 345 fully evaluable MM IGH sequences. This database was then used to comprehensively investigate the characteristics of the IGH rearrangement in MM. == Design and Methods == == Collection of immunoglobulin sequences == The original database collected for this study consisted of 406 MM IGH sequences. A total of 237 MM IGH sequences were retrieved from EMBL, NCBI and IMGT/LIGM-DB public databases, as already reported (Literature Series, LS),10,11,14while 169 were directly sequenced at our institutions for MRD purposes (Institutional Series, Is usually). All patients provided informed consent in accordance with the requirements of the local Institutional Review Board (IRB) and with the Declaration of Helsinki. The study was approved by the IRB Commissione Regionale per le Sperimentazioni Cliniche, Turin, Italy. Methods for IGH sequencing have been described elsewhere.19Redundant, poorly annotated, out-of-frame, or clonally related sequences were excluded from the analysis, as previously reported.10,11,14Therefore, the final collection included 345 evaluable IGH MM sequences (MM total, each sequence corresponding to one single patient), 214 from LS (Online Supplementary Table S1) and 131 from IS (Online Supplementary Table S2). For clustering analysis, a total of 28,376 non-MM IGH sequences were retrieved from public databases (n=26,618)10,11,14and from our multi-laboratory database (n=1,758). The non-MM cohort intentionally included several different entities to allow comparison with various types of B cells (Online Supplementary Table S3). In particular, a subanalysis was also performed on 461 sequences from PC from healthy donors. == Sequence analysis and data mining == All sequences were aligned to the IMGT directory site for the identification of Splenopentin Acetate IGH rearrangements, following established criteria.10,11,20IGHV,IGHD, andIGHJgene usage, somatic hypermutation (SHM) load, and the complementarity determining region 3 on heavy chain gene (HCDR3) length were recorded for each sequence. Comparisons in terms ofIGHVusage were made between the MM series and the largest published series ofIGHVsequences from healthy donors.10Only smaller comparative series from healthy donors were available to compareIGHD-Jusage, mutational load, and HCDR3 length distribution.10,2126 == HCDR3-driven clustering == Clustering analysis was performed using ClustalX 2.0, as previously described.10,27Stereotyped HCDR3 sequences were those characterized by an aminoacidic identity of 60% or over, according to the criteria of Messmer and Stamatopoulos.10,28Subsets not previously described and including only two sequences (provisional) were SX-3228 only considered if they fulfilled the following additional criteria:11,12,14i) use ofIGHVgermline genes of the same clan; ii) use of the sameIGHDandIGHJgermline genes; iii) use of the sameIGHDsegment reading frame; and iv) identical HCDR3 length. == Statistical analysis == Patients characteristics were tested using Fishers exact test for discrete variables and the Mann-Whitney test for continuous variables. All reportedPvalues were obtained by a two-sided exact method, at the conventional 5% significance level. Data were analyzed.